Gold ion beam drilled nanopores modified with thiolated DNA origamis

ABSTRACT

A nanopore structure includes an aperture extending from a first surface to a second surface of a substrate, the aperture having a wall comprising gold ions embedded in the substrate, the wall defining a first diameter; a first deoxyribonucleic acid (DNA) layer including a thiolated DNA strand covalently bonded to the embedded gold ions within the wall of the aperture; and a second DNA layer hydrogen bonded to the first DNA layer, the second DNA layer defines a substantially cylindrical nanopore that defines a second diameter within the wall of the aperture, the second DNA layer including a single-stranded DNA strand; wherein the second diameter is less than the first diameter.

BACKGROUND

The present disclosure generally relates to nanopores, and morespecifically, to artificial nanopores.

Nanopore development for single molecule sensing applications is arapidly growing field. Natural nanopores are gene-expressed andgenerally form an aperture, or channel, between two lipid membranes.Various molecules, for example, proteins, deoxyribonucleic acid (DNA),and other small molecules, traverse membranes through the nanopore. Whena potential difference is generated across the lipid bilayer, ioniccurrent can be monitored. A change in ionic current can indicate that amolecule(s) is binding to and/or moving through the nanopore. Differenttypes of molecules can demonstrate different ionic current changes.

Natural or biological nanopores, such as alpha-hemolysin, can detect andidentify DNA bases for sequencing applications. These nanopores also canbe used for detecting drugs, explosives, or chemical warfare agents atthe single molecule level.

Protein nanopores are nanopores that are genetically engineered torecognize different molecules, for example, different DNA bases. Thesegenetically engineered nanopores can provide data with relatively highsignals and resolutions. Like natural nanopores, however, geneticallyengineered nanopores rely on a suspended lipid bilayer for deviceoperation.

Artificial nanopores do not rely on a lipid membrane. Artificialnanopores can include, for example, silicon nitride (SiN) or siliconoxide (SiO) and are generally apertures through two solid surfaces.

SUMMARY

In one embodiment of the present disclosure, a nanopore structureincludes an aperture extending from a first surface to a second surfaceof a substrate, the aperture having a wall including gold ions embeddedin the substrate, the wall defining a first diameter; a firstdeoxyribonucleic acid (DNA) layer comprising a thiolated DNA strandcovalently bonded to the embedded gold ions within the wall of theaperture; and a second DNA layer hydrogen bonded to the first DNA layer,the second DNA layer defines a substantially cylindrical nanopore thatdefines a second diameter within the wall of the aperture, the secondDNA layer including a single-stranded DNA strand; wherein the seconddiameter is less than the first diameter.

In another embodiment, a nanopore structure includes an apertureextending from a first surface to a second surface of a substrate, theaperture having a wall including gold ions embedded in the substrate,the wall defining a first diameter; a first deoxyribonucleic acid (DNA)layer comprising a thiolated DNA strand covalently bonded to theembedded gold ions within the wall of the aperture; a second DNA layerhydrogen bonded to the first DNA layer, the second DNA layer defines asubstantially cylindrical nanopore that defines a second diameter withinthe wall of the aperture, the second DNA layer including asingle-stranded DNA strand; and a biomolecule binding site on thesingle-stranded DNA strand, the biomolecule binding site being achemical functional group that is chemically bonded to thesingle-stranded DNA strand or a DNA sequence within the single-strandedDNA strand; wherein the second diameter is less than the first diameter.

Yet, in another embodiment, a method for making a nanopore structureincludes drilling with a gold ion beam to define an aperture through asubstrate and to introduce and embed gold ions within a wall of theaperture, the wall defining a first diameter; bonding a thiolated DNAstrand to the embedded gold ions with a covalent bond; and hydrogenbonding a single-stranded DNA strand to the thiolated DNA strand with acovalent bond to define a substantially cylindrical nanopore, thesubstantially cylindrical nanopore defining a second diameter within theaperture; wherein the second diameter is less than the first diameter.

BRIEF DESCRIPTION OF THE DRAWINGS

The subject matter which is regarded as the invention is particularlypointed out and distinctly claimed in the claims at the conclusion ofthe specification. The forgoing and other features, and advantages ofthe invention are apparent from the following detailed description takenin conjunction with the accompanying drawings in which:

FIG. 1A illustrates a cross-sectional side view of a substrate beforegold ion beam drilling;

FIG. 1B illustrates a cross-sectional side view of an aperture extendingthrough the substrate of FIG. 1B after gold ion beam drilling;

FIG. 2A illustrates a cross-sectional side view of thiolated DNA boundto gold ions within the aperture of FIG. 1A;

FIG. 2B illustrates a top view of FIG. 2A;

FIG. 3A illustrates a cross-sectional side view of single-stranded DNAhybridized to the thiolated DNA of FIG. 2A;

FIG. 3B illustrates a top view of FIG. 3A;

FIG. 4A illustrates a cross-sectional side view of biomolecules bound tothe single-stranded DNA of FIG. 3A;

FIG. 4B illustrates a top view of FIG. 4A;

FIGS. 5A, 5B, and 5C respectively illustrate top views of substantiallycylindrical nanopores having substantially the same inner diameterswithin apertures of different diameters;

FIG. 6 shows a dose test array membrane milled with Au²⁺ ions at 2.3picoÅmperes (pÅ) with varying dwell times; and

FIG. 7 shows a single pore fabricated by a Au²⁺ beam with a 4.5 ms dwelltime at a 2.3 pÅ beam current.

DETAILED DESCRIPTION

Artificial nanopore development is lagging behind protein poredevelopment. One reason is because artificial nanopores with biologicalnanopore dimensions are challenging to produce. Also, the control overprecise structure and chemical functionalities involved in singlemolecule recognition and interaction are difficult in artificialnanopores. Further, while biological nanopores are genetically designedto position a key chemical feature in a precise location, artificialpores are drilled through a material. Thus, artificial nanopores mayresult in varying shapes and surface chemistries inside the nanopores,with little or no control over such parameters.

Accordingly, the present disclosure solves the above problems bycreating a chemical interface inside an artificial nanopore. Theartificial nanopore surface interior includes self-assembledthree-dimensional (3D) structures. The surface structures create ascaffold within the nanopore with specific binding sites for chemicalfunctionalities in specific positions within the artificial pore. Thedisclosed artificial nanopores restore control over the chemicalstructure of the nanopore inner surface.

As stated above, the present disclosure relates to nanopores, andparticularly to artificial nanopores, which are now described in detailwith accompanying figures. It is noted that like reference numeralsrefer to like elements across different embodiments.

The following definitions and abbreviations are to be used for theinterpretation of the claims and the specification. As used herein, theterms “comprises,” “comprising,” “includes,” “including,” “has,”“having,” “contains” or “containing,” or any other variation thereof,are intended to cover a non-exclusive inclusion. For example, acomposition, a mixture, process, method, article, or apparatus thatcomprises a list of elements is not necessarily limited to only thoseelements but can include other elements not expressly listed or inherentto such composition, mixture, process, method, article, or apparatus.

As used herein, the articles “a” and “an” preceding an element orcomponent are intended to be nonrestrictive regarding the number ofinstances (i.e. occurrences) of the element or component. Therefore, “a”or “an” should be read to include one or at least one, and the singularword form of the element or component also includes the plural unlessthe number is obviously meant to be singular.

As used herein, the terms “invention” or “present invention” arenon-limiting terms and not intended to refer to any single aspect of theparticular invention but encompass all possible aspects as described inthe specification and the claims.

As used herein, the term “about” modifying the quantity of aningredient, component, or reactant of the invention employed refers tovariation in the numerical quantity that can occur, for example, throughtypical measuring and liquid handling procedures used for makingconcentrates or solutions. Furthermore, variation can occur frominadvertent error in measuring procedures, differences in themanufacture, source, or purity of the ingredients employed to make thecompositions or carry out the methods, and the like. In one aspect, theterm “about” means within 10% of the reported numerical value. Inanother aspect, the term “about” means within 5% of the reportednumerical value. Yet, in another aspect, the term “about” means within10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of the reported numerical value.

Referring now to the figures, FIG. 1A illustrates a cross-sectional sideview of a nanopore structure's substrate 110 before drilling with a goldion beam 120. The substrate has a first surface 112 and a second surface114 opposite the first surface 112.

Non-limiting examples of suitable substrate materials include silicon,silicon oxide, silicon nitride, or any combination thereof. Thesubstrate 110 can include a single layer or multiple layers of any ofthe aforementioned materials or layers or any additional materials andlayers.

The substrate 110 may be fabricated using any suitable fabricationprocess. Non-limiting examples of suitable fabrication processesinclude, for example, chemical vapor deposition (CVD), plasma enhancedchemical vapor deposition (PECVD), lithographic patterning and etching,epitaxial growth processes, or any combination thereof.

The thickness of the substrate 110 is not intended to be limited. In oneembodiment, the substrate 110 has a thickness in a range from about 10to about 100 nanometers (nm). In another embodiment, the substrate 110has a thickness in a range from about 20 to about 50 nm. Yet, in otherembodiments, the substrate 110 has a thickness about or in any rangefrom about 10, 25, 50, 75, 100, 125, and 150 nm.

The drilling with a gold ion beam 120 may be focused ion beam (FIB) oranother suitable method. A FIB setup uses a focused beam of gold ionsfor deposition or ablation of materials.

FIG. 1B illustrates a cross-sectional side view of a nanopore structurehaving an aperture 140 extending through the substrate 110 of FIG. 1Aafter drilling with a gold ion beam 120. The aperture 140 extends fromthe first surface 112 to the second surface 114.

Drilling with a gold ion beam 120 uses a gold ion beam to drill theaperture 140. Gold ions 130 (Au²⁺) heavily contaminate the wall 150 or aportion of the wall 150 of the aperture 140. Gold ions 130 are embeddedin the substrate 110 in a region confined to the walls of the aperture140. The remaining regions of the substrate 110 are substantially freeof gold ions. Gold ions are used to create a covalent bound with thethiolated DNA.

The gold ion beam patterning parameters can be adjusted to form anaperture 140 with the desired diameter within the substrate 110. Forexample, the acceleration voltage, beam current, dwell time per pointcan be varied. Increasing the dwell time and beam current can increasethe diameter of the apertures 140.

The thickness, t, of the embedded gold ions 130 within the substrate 110can generally vary. In some embodiments, the thickness t of the embeddedgold ions 130 is in a range from about 2 to about 40 nm. In oneembodiment, the thickness t of the embedded gold ions 130 is in a rangefrom about 5 to about 10 nm. In other embodiments, the thickness t ofthe embedded gold ions 130 is in a range about or in any range fromabout 2, 5, 10, 15, 20, 25, 30, 35, and 40 nm.

The wall 150 of the aperture 140 has a diameter d1 (a first diameter)that can generally vary and is not intended to be limited. In oneembodiment, the diameter d1 is in a range from about 10 to about 100. Inone embodiment, the diameter d1 is in a range from about 20 to about 40.In other embodiments, the diameter d1 is in a range about or in anyrange from about 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 nm.

FIG. 2A illustrates a cross-sectional side view of a first DNA layerbound to gold ions 130 within the wall 150 of the aperture 140 of FIG.1A. FIG. 2B illustrates a top view of FIG. 2A. The first DNA layerincludes a thiolated DNA strand 210 chemically bonded to gold ions 130within the wall 150 of the aperture 140. The thiolated DNA strand 210includes a thiolate group that covalently bonds to the gold ions 130.The thiolated DNA 210 is a single-stranded DNA strand. One or morethiolated DNA strands 210 binds to the gold ions 130 within the aperture140. The thiolated DNA strand 210 dangles within the wall 150 aperture140 and forms an anchor within the aperture 140.

Each thiolated DNA strand 210 has a length that can generally vary. Inone embodiment, each thiolated DNA strand 210 has a length in a rangefrom about 30 to about 200 bases. In another embodiment, each thiolatedDNA strand 210 has a length in a range from about 60 to about 110 bases.

FIG. 3A illustrates a cross-sectional side view of a second DNA layerchemically bonded to the first DNA layer of FIG. 2A via hydrogen bonds.The second DNA layer includes a single-stranded DNA strand 310 that ishydrogen bonded to the thiolated DNA strand 210. FIG. 3B illustrates atop view of FIG. 3A. The single-stranded DNA strand 310 is hybridized(bonded via hydrogen bonds) to the thiolated DNA 210 such that thesingle-stranded DNA strand 310 defines a substantially cylindricalnanopore within the aperture 140. The single-stranded DNA strand 310includes biofunctional group binding sites 320 to biofunctionalize thesubstantially cylindrical nanopore.

The binding sites 320 can be any functional group, chemicalfunctionality, or specific DNA sequence within the single-stranded DNA310 where biomolecules 410 can be attached. Non-limiting examples ofsuitable chemical functional groups include alcohols, amines,carboxylates, esters, ethers, amides, thiols, aldehydes, ketones, acylhalides, nitriles, imines, isocyanates, azo compounds, arenes, acidanhydrides, alkanes, alkenes, alkynes, or any combination thereof. Thefunctional groups may be substituted or non-substituted. The functionalgroups may be charged or un-charged.

The single-stranded DNA strand 310 forms a 3D DNA origami frame insidethe aperture 140. As described herein, a 3D DNA origami is the nanoscalefolding of DNA to create arbitrary two and three-dimensional shapes atthe nanoscale. The DNA origami frame can remain inside the aperture 140even if the thiolated DNA strand 210 anchor molecules detach from thegold ions 130 embedded in the wall 150 of the aperture 140. Theresulting DNA origami frame provides a structure with sequence specificanchor points where specific functionalities can be preciselypositioned.

The substantially cylindrical nanopore has a diameter d2 (a seconddiameter) (see FIG. 3B) that can generally vary and is not intended tobe limited. The diameter d2 is less than the diameter d1 of the aperture(the first diameter). In one embodiment, the diameter d2 is in a rangefrom about 5 to about 30 nm. In another embodiment, the diameter d2 isin a range from about 5 to about 15 nm. In other embodiments, thediameter d2 is in a range about or in any range from about 5, 10, 15,20, 25, and 30 nm.

FIG. 4A illustrates a cross-sectional side view of biomolecules 410bound to the single-stranded DNA strands 310 of FIG. 3A. FIG. 4Billustrates a top view of FIG. 4A. Biomolecules 410 will bind to thebiofunctional group binding sites 320. The biomolecules 410 attach tothe DNA origami scaffold formed by the single-stranded DNA stand 310 toform a protein-like inner nanopore. The biomolecules 410 provide forwetting and affinity with target analytes. The biomolecules 410 can bechemically bonded to the binding sites 320.

The biomolecules 410 can be any molecule present in living organisms.Non-limiting examples of suitable biomolecules 410 macromolecules, forexample, proteins, polysaccharides, lipids, and nucleic acids, and smallmolecules, for example, primary metabolites, secondary metabolites, andnatural products. Other small molecules include, but are not limited to,lipids, fatty acids, glycolipids, sterols, glycerolipids, vitamins,hormones, neurotransmitters, metabolites, monomers, oligomers, polymers,or any combination thereof. The biomolecules 410 can be biomonomers, forexample, amino acids, monosaccharides, or nucleotides. The biomolecules410 can be bio-oligomers, for example, oligopeptides, oligosaccharides,or oligonucleotides. Other non-limiting examples of suitablebiomolecules include DNA, ribonucleic acid (RNA), glyco lipids,phospholipids, and enzymes.

FIGS. 5A, 5B, and 5C illustrate top views of substantially cylindricalnanopores within apertures 140 having different diameters. Each aperture140 has a first diameter d1(a), d1(b), and d1(c) that generally varies,while the substantially cylindrical nanopore within the aperture 140 hasa substantially fixed second diameter d2.

In the exemplary embodiments of FIGS. 5A-5C, the first diametersdecrease in size as follows: d1(a)>d1(b)>d1(c). Simultaneously, thesecond diameters d2 are substantially the same. The thiolated DNA 210changes length by extending or curling to accommodate for variations inthe gold ion drilled aperture diameter d1(a), d1(b), and d1(c). Thethiolated DNA 210 has a variable length that depends on the aperture's140 first diameter d1 and such that the substantially cylindricalnanopore's second diameter d2 is substantially fixed.

Thus substantially cylindrical nanopores, which mirror proteinnanopores, can be fitted within an existing artificial nanopore. Theapproach leaves gaps between the DNA outer structure formed by thethiolated DNA strands 210 and the artificial nanopore structure within,formed by the single-stranded DNA 410.

The disclosed nanopore structures solve the problem caused by variableartificial nanopore structure dimensions, as shown in FIGS. 5A-5C, thatresult in non-reproducible results. Small variations in artificialnanopore dimensions caused by manufacturing variability and artifactsbecome more problematic as nanopores become smaller. By forming the DNAnanopore out of the inner structure of the artificial 3D DNA nanopore,specific nanopores with consistent dimensions provide reliable andreproducible results.

EXAMPLE

To demonstrate the capability of gold ion beam lithography for millingof nanopores in thin membranes, membrane chips were processed using theRaith ionLiNE system (Raith Nano fabrication, Dortmund, Germany). About100 membranes were ion beam drilled using the Raith multi speciesdevelopment tool.

The patterning parameters were as follows: acceleration voltage 40 kV;beam current 1.4-3.3 pÅ; and dwell time per point 2.5-60 ms.

FIG. 6 shows a dose test array membrane milled with Au²⁺ ions at 2.3picoÅmperes (pÅ). Various dwell times were tested, including 3.5 ms,3.65 ms, and 4 ms. The resulting apertures had exemplary diameters of12.21 nm, 15.63 nm, and 16.75 nm.

FIG. 7 shows a single pore fabricated by a Au²⁺ beam with a 4.5 ms dwelltime at a 2.3 pÅ beam current. The resulting aperture diameter was 12.67nm.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, element components,and/or groups thereof.

The corresponding structures, materials, acts, and equivalents of allmeans or step plus function elements in the claims below are intended toinclude any structure, material, or act for performing the function incombination with other claimed elements as specifically claimed. Thedescription of the present invention has been presented for purposes ofillustration and description, but is not intended to be exhaustive orlimited to the invention in the form disclosed. Many modifications andvariations will be apparent to those of ordinary skill in the artwithout departing from the scope and spirit of the invention. Theembodiment was chosen and described in order to best explain theprinciples of the invention and the practical application, and to enableothers of ordinary skill in the art to understand the invention forvarious embodiments with various modifications as are suited to theparticular use contemplated.

The flow diagrams depicted herein are just one example. There may bemany variations to this diagram or the steps (or operations) describedtherein without departing from the spirit of the invention. Forinstance, the steps may be performed in a differing order or steps maybe added, deleted or modified. All of these variations are considered apart of the claimed invention.

The descriptions of the various embodiments of the present inventionhave been presented for purposes of illustration, but are not intendedto be exhaustive or limited to the embodiments disclosed. Manymodifications and variations will be apparent to those of ordinary skillin the art without departing from the scope and spirit of the describedembodiments. The terminology used herein was chosen to best explain theprinciples of the embodiments, the practical application or technicalimprovement over technologies found in the marketplace, or to enableothers of ordinary skill in the art to understand the embodimentsdisclosed herein.

What is claimed is:
 1. A nanopore structure, comprising: an apertureextending from a first surface to a second surface of a substrate, theaperture having a wall extending from the first surface to the secondsurface comprising gold ions embedded in the substrate and wherein thegold ions are in a region confined to the walls of the aperture, thewall defining a first diameter; a first deoxyribonucleic acid (DNA)layer comprising a thiolated DNA strand covalently bonded to theembedded gold ions within the wall of the aperture; and a second DNAlayer hydrogen bonded to the first DNA layer, the second DNA layerdefines a substantially cylindrical nanopore that defines a seconddiameter within the wall of the aperture, the second DNA layercomprising a single-stranded DNA strand; wherein the second diameter isless than the first diameter; and wherein a thickness of the embeddedgold ions within the wall of the aperture is in a range from about 2 toabout 40 nm.
 2. The nanopore structure of claim 1, the single-strandedDNA strand further comprising a binding site to biofunctionalize thesubstantially cylindrical nanopore.
 3. The nanopore structure of claim1, further comprising a biomolecule chemically bonded to the bindingsite.
 4. The nanopore structure of claim 1, wherein the substratecomprises silicon, silicon oxide, silicon nitride, or any combinationthereof.
 5. The nanopore structure of claim 1, wherein the firstdiameter is in a range from about 10 to about 100 nanometers (nm). 6.The nanopore structure of claim 1, wherein the second diameter is in arange from about 5 to about 30 nm.
 7. A nanopore structure, comprising:an aperture extending from a first surface to a second surface of asubstrate, the aperture having a wall extending from the first surfaceto the second surface comprising gold ions embedded in the substrate andwherein the gold ions are in a region confined to the walls of theaperture, the wall defining a first diameter; a first deoxyribonucleicacid (DNA) layer comprising a thiolated DNA strand covalently bonded tothe embedded gold ions within the wall of the aperture; a second DNAlayer hydrogen bonded to the first DNA layer, the second DNA layerdefines a substantially cylindrical nanopore that defines a seconddiameter within the wall of the aperture, the second DNA layercomprising a single-stranded DNA strand; and a biomolecule binding siteon the single-stranded DNA strand, the biomolecule binding site being achemical functional group that is chemically bonded to thesingle-stranded DNA strand or a DNA sequence within the single-strandedDNA strand; wherein the second diameter is less than the first diameter;and wherein a thickness of the embedded gold ions within the wall of theaperture is in a range from about 2 to about 40 nm.
 8. The nanoporestructure of claim 7, wherein the thiolated DNA strand comprises athiolate group that forms a covalent bond with the embedded gold ionswithin the wall of the aperture.
 9. The nanopore structure of claim 7,wherein the substrate comprises silicon, silicon oxide, silicon nitride,or any combination thereof.
 10. The nanopore structure of claim 7,wherein the thiolated DNA strand has a variable length that depends onthe first diameter.
 11. The nanopore structure of claim 10, wherein thesecond diameter is substantially fixed.
 12. A nanopore structure,comprising: an aperture extending from a first surface to a secondsurface of a substrate, the aperture having a wall extending from thefirst surface to the second surface comprising gold ions embedded in thesubstrate and wherein the gold ions are in a region confined to thewalls of the aperture, the wall defining a first diameter; a firstdeoxyribonucleic acid (DNA) layer comprising a plurality ofsingle-stranded thiolated DNA strands, the plurality of single-strandedthiolated DNA strands of the first DNA layer covalently bonded to theembedded gold ions through a thiolate group, and each single-strandedthiolated DNA strand extending and dangling within the wall of theaperture to form an anchor within the aperture; a second DNA layercomprising a plurality of single-stranded DNA strands, the plurality ofsingle-stranded DNA strands of the second DNA layer hybridized to thefirst DNA layer through hydrogen bonds, the second DNA layer defines asubstantially cylindrical nanopore that defines a second diameter withinthe wall of the aperture hydrogen bonded to the first DNA layer, thesecond DNA layer defines a substantially cylindrical nanopore thatdefines a second diameter within the wall of the aperture, the secondDNA layer comprising a single-stranded DNA strand; and a biomoleculebinding site on the second DNA layer, the biomolecule binding site beinga DNA sequence within the single-stranded DNA strand; wherein the seconddiameter is less than the first diameter, and each single-strandedthiolated DNA strand of the first DNA layer has a variable length thatdepends on the first diameter of the wall of the aperture such that thesecond diameter of the substantially cylindrical nanopore issubstantially fixed; and wherein a thickness of the embedded gold ionswithin the wall of the aperture is in a range from about 2 to about 40nm.